Minimum procedure system for the determination of analytes

ABSTRACT

A method for determining the presence of an analyte in a fluid is described along with various components of an apparatus specifically designed to carry out the method. The method involves taking a reflectance reading from one surface of an inert porous matrix impregnated with a reagent that will interact with the analyte to produce a light-absorbing reaction product when the fluid being analyzed is applied to another surface and migrates through the matrix to the surface being read. Reflectance measurements are made at two separate wavelengths in order to eliminate interferences, and a timing circuit is triggered by an initial decrease in reflectance by the wetting of the surface whose reflectance is being measured by the fluid which passes through the inert matrix. The method and apparatus are particularly suitable for the measurement of glucose levels in blood without requiring separation of red blood cells from serum or plasma.

FIELD OF THE INVENTION

[0001] The present invention relates to a test device and method for thecalorimetric determination of chemical and biochemical components(analytes) in aqueous fluids, particularly whole blood. In one preferredembodiment it concerns a test device and method for calorimetricallymeasuring the concentration of glucose in whole blood.

BACKGROUND OF THE INVENTION

[0002] The quantification of chemical and biochemical components incolored aqueous fluids, in particular colored biological fluids such aswhole blood and urine and biological fluid derivatives such as bloodserum and blood plasma, is of ever-increasing importance. Importantapplications exist in medical diagnosis and treatment and in thequantification of exposure to therapeutic drugs, intoxicants, hazardouschemicals and the like. In some instances, the amounts of materialsbeing determined are either so miniscule—in the range of a microgram orless per deciliter—or so difficult to precisely determine that theapparatus employed is complicated and useful only to skilled laboratorypersonnel. In this case the results are generally not available for somehours or days after sampling. In other instances, there is often anemphasis on the ability of lay operators to perform the test routinely,quickly and reproducibly outside a laboratory setting with rapid orimmediate information display.

[0003] One common medical test is the measurement of blood glucoselevels by diabetics. Current teaching counsels diabetic patients tomeasure their blood glucose level from two to seven times a daydepending on the nature and severity of their individual cases. Based onthe observed pattern in the measured glucose levels the patient andphysician together make adjustments in diet, exercise and insulin intaketo better manage the disease. Clearly, this information should beavailable to the patient immediately.

[0004] Currently a method widely used in the United States employs atest article of the type described in U.S. Pat. No. 3,298,789 issuedJan. 17, 1967 to Mast. In this method a sample of fresh, whole blood(typically 20-40 μl) is placed on an ethylcellulose-coated reagent padcontaining an enzyme system having glucose oxidase and peroxidaseactivity. The enzyme system reacts with glucose and releases hydrogenperoxide. The pad also contains an indicator which reacts with thehydrogen peroxide in the presence of peroxidase to give a colorproportional in intensity to the sample's glucose level.

[0005] Another popular blood glucose test method employs similarchemistry but in place of the ethylcellulose-coated pad employs awater-resistant film through which the enzymes and indicator aredispersed. This type of system is disclosed in U.S. Pat. No. 3,630,957issued Dec. 28, 1971 to Rey et al.

[0006] In both cases the sample is allowed to remain in contact with thereagent pad for a specified time (typically one minute). Then in thefirst case the blood sample is washed off with a stream of water whilein the second case it is wiped off the film. The reagent pad or film isthen blotted dry and evaluated. The evaluation is made either bycomparing color generated with a color chart or by placing the pad orfilm in a diffuse reflectance instrument to read a color intensityvalue.

[0007] The presence of red blood cells or other colored components ofteninterferes with the measurements of these absolute values therebycalling for exclusion of red blood cells in these two prior methods asthey are most widely practiced. In the device of U.S. Pat. No. 3,298,789an ethyl cellulose membrane prevents red blood cells from entering thereagent pad. Similarly, the water-resistant film of U.S. Pat. No.3,630,957 prevents red blood cells from entering. In both cases therinse or wipe also acts to remove these potentially interfering redblood cells prior to measurement.

[0008] Accordingly, there remains a need for a system of detectinganalytes in colored liquids, such as blood, that does not requireremoval of excess liquid from a reflectance strip from which areflectance reading is being obtained.

SUMMARY OF THE INVENTION

[0009] Novel methods, compositions and apparatus are provided fordiagnostic assays comprising a hydrophilic porous matrix containing asignal producing system and a reflectance measuring apparatus which isactivated upon a change in reflectance of the matrix when fluidpenetrates the matrix. The method comprises adding the sample, typicallywhole blood, to the matrix which filters out large particles, such asred blood cells, typically with the matrix present in the apparatus. Thesignal-producing system produces a product which further changes thereflectance of the matrix, which change can be related to the presenceof an analyste in a sample.

[0010] Exemplary of the diagnostic assay system is the determination ofglucose in the whole blood, where the determination is made withoutinterference from the blood and without a complicated protocol subjectto use error.

BRIEF DESCRIPTION OF THE DRAWINGS

[0011] The present invention can be more readily understood by referenceto the following detailed description when read in conjunction with theattached drawings, wherein:

[0012]FIG. 1 is a perspective view of one embodiment of a test devicecontaining the reaction pad to which the fluid being analyzed isapplied.

[0013]FIG. 2 is a block diagram schematic of an apparatus that can beemployed in the practice of the invention.

[0014]FIG. 3 is a block diagram schematic of an alternate apparatus thatcan be employed in the practice of the invention.

DETAILED DESCRIPTION OF THE INVENTION The Reagent Element

[0015] The subject invention provides an improved rapid and simplemethodology employing reliable and easy to operate apparatus for thedetermination of analytes such as glucose, particularly involving anenzyme substrate which results in the production of hydrogen peroxide asan enzyme product. The method involves applying to a porous matrix asmall volume of whole blood, sufficient to saturate the matrix. Bound tothe matrix are one or more reagents of a signal producing system, whichresults in the production of a product resulting an initial change inthe amount of reflectance of the matrix. The matrix is typically presentin a reflectance-measuring apparatus when blood is applied. The liquidsample penetrates the matrix, resulting in an initial change inreflectance at the measurement surface. A reading is then taken at oneor more times after the initial change in reflectance to relate thefurther change in reflectance at the measurement surface or in thematrix as a result of formation of the reaction product to the amount ofanalyte in the sample.

[0016] For measurements in blood, particularly glucose measurements,whole blood is typically used as the assay medium. The matrix containsan oxidase enzyme which produces hydrogen peroxide. Also contained inthe matrix will be a second enzyme, particularly a peroxidase, and a dyesystem which produces a light-absorbing product in conjunction with theperoxidase. The light-absorbing product changes the reflectance signal.With whole blood, readings are taken at two different wavelengths withthe reading at one wavelength used to subtract out backgroundinterference caused by hematocrit, blood oxygenation, and othervariables which may affect the result.

[0017] A reagent element is employed which comprises the matrix and themembers of the signal producing system contained within the matrix. Thereagent element may include other components for particularapplications. The method requires applying a small volume of blood,which typically has not been subject to prior treatment (other thanoptional treatment with an anticoagulant), to the matrix. Timing of themeasurement is activated by the apparatus automatically detecting achange in reflectance of the matrix when fluid penetrates the matrix.The change in reflectance over a predetermined time period as a resultof formation of reaction product is then related to the amount ofanalyte in a sample.

[0018] The first component of the present invention to be considered isa reagent element, conveniently in the shape of a pad, comprising aninert porous matrix and the component or components of asignal-producing system, which system is capable of reacting with ananalyte to produce a light-absorbing reaction product, impregnated intothe pores of the porous matrix. The signal-producing system does notsignificantly impede the flow of liquid through the matrix.

[0019] In order to assist in reading reflectance, it is preferred thatthe matrix have at least one side which is substantially smooth andflat. Typically, the matrix will be formed into a thin sheet with atleast one smooth, flat side. In use, the liquid sample being analyzed isapplied to one side of the sheet whereby any assay compound presentpasses through the reagent element by means of capillary, wicking,gravity flow and/or diffusion actions. The components of the signalproducing system present in the matrix will react to give a lightabsorbing reaction product. Incident light impinges upon the reagentelement at a location other than the location to which the sample isapplied. Light is reflected from the surface of the element as diffusereflected light. This diffuse light is collected and measured, forexample by the detector of a reflectance spectrophotometer. The amountof reflected light will be related to the amount of analyte in thesample, usually being an inverse function of the amount of analyte inthe sample.

The Matrix

[0020] Each of the components necessary for producing the reagentelement will be described in turn. The first component is the matrixitself.

[0021] The matrix will be a hydrophilic porous matrix to which reagentsmay be covalently or non-covalently bound. The matrix will allow for theflow of an aqueous medium through the matrix. It will also allow forbinding of protein compositions to the matrix without significantlyadversely affecting the biological activity of the protein, e.g.enzymatic activity of an enzyme. To the extent that proteins are to becovalently bound, the matrix will have active sites for covalent bondingor may be activated by means known to the art. The composition of thematrix will be reflective and will be of sufficient thickness to permitthe formation of a light-absorbing dye in the void volume or on thesurface to substantially affect the reflectance from the matrix. Thematrix may be of a uniform composition or a coating on a substrateproviding the necessary structure and physical properties.

[0022] The matrix will usually not deform on wetting, thus retaining itsoriginal conformation and size. The matrix will have a definedabsorbtivity, so that the volume which is absorbed can be calibratedwithin reasonable limits, variations usually being maintained belowabout 50%, preferably not greater than 10%. The matrix will havesufficient wet strength to allow for routine manufacture. The matrixwill permit non-covalently bound reagents to be relatively uniformlydistributed on the surface of the matrix.

[0023] As exemplary of matrix surfaces are polyamides, particularly withsamples involving whole blood. The polyamides are convenientlycondensation polymers of monomers of from 4 to 8 carbon atoms, where themonomers are lactams or combinations of diamines and di-carboxylicacids. Other polymeric compositions having comparable properties mayalso find use. The polyamide compositions may be modified to introduceother functional groups which provide for charged structures, so thatthe surfaces of the matrix may be neutral, positive or negative, as wellas neutral, basic or acidic. Preferred surfaces are positively charged.

[0024] When used with whole blood, the porous matrix preferably haspores with an average diameter in the range of from about 0.1 to 2.0 μm,more preferrably from about 0.6 to 1.0 μm.

[0025] A preferred manner of preparing the porous material is to castthe hydrophilic polymer onto a core of non-woven fibers. The core fiberscan be any fibrous material that produce the described integrity andstrength, such as polyesters and polyamides. The reagent that will formthe light-absorbing reaction product, which is discussed later indetail, is present within the pores of the matrix but does not block thematrix so that the liquid portion of the assay medium, e.g. blood, beinganalyzed can flow through the pores of the matrix, while particles, suchas erythrocytes, are held at the surface.

[0026] The matrix is substantially reflective so that it gives a diffusereflectance without the use of a reflective backing. Preferably at least25%, more preferably at least 50%, of the incident light applied to thematrix is reflected and emitted as diffuse reflectance. A matrix of lessthan about 0.5 mm thickness is usually employed, with from about 0.01 to0.3 mm being preferred. A thickness of from 0.1 to 0.2mm is mostpreferred, particularly for a nylon matrix.

[0027] Typically, the matrix will be attached to a holder in order togive it physical form and rigidity, although this may not be necessary.FIG. 1 shows one embodiment of the invention in which a thin hydrophilicmatrix pad 11 is positioned at one end of a plastic holder 12 by meansof an adhesive 13 which directly and firmly attaches the reagent pad tothe handle. A hole 14 is present in the plastic holder 12 in the area towhich reagent pad 11 is attached so that sample can be applied to oneside of the reagent pad and light reflected from the other side.

[0028] A liquid sample to be tested is applied to pad 11. Generally,with blood being exemplary of a sample being tested, the reagent padwill be on the order of about 10 mm² to 100 mm² in surface area,especially 10 mm² to 50 mm² in area, which is normally a volume that5-10 microliters of sample will more than saturate.

[0029] Diffuse reflectance measurements in the prior art have typicallybeen taken using a reflective backing attached to or placed behind thematrix. No such backing is needed or will normally be present during thepractice of the present invention, either as part of the reagent elementor the reflectance apparatus.

[0030] As can be seen from FIG. 1, the support holds reagent pad 11 sothat a sample can be applied to one side of the reagent pad while lightreflectance is measured from the side of the reagent pad opposite thelocation where sample is applied.

[0031]FIG. 2 shows a system in which the reagent is applied to the sidewith the hole in the backing handle while light is reflected andmeasured on the other side of the reagent pad. Other structures than theone depicted may be employed. The pad may take various shapes and forms,subject to the limitations provided herein. The pad will be accessibleon at least one surface and usually two surfaces.

[0032] The hydrophilic layer (reagent element) may be attached to thesupport by any convenient means, e.g., a holder, clamp or adhesives;however, in the preferred method it is bonded to the backing. Thebonding can be done with any non-reactive adhesive, by a thermal methodin which the backing surface is melted enough to entrap some of thematerial used for the hydrophilic layer, or by microwave or ultrasonicbonding methods which likewise fuse the hydrophilic sample pads to thebacking. It is important that the bonding be such as to not itselfinterfere substantially with the diffuse reflectance measurements or thereaction being measured, although this is unlikely to occur as noadhesive need be present at the location where the reading is taken. Forexample, an adhesive 13 can be applied to the backing strip 12 followedfirst by punching hole 14 into the combined strip and adhesive and thenapplying reagent pad 11 to the adhesive in the vicinity of hole 14 sothat the peripheral portion of the reagent pad attaches to the backingstrip.

The Chemical Reagents

[0033] Any signal producing system may be employed that is capable ofreacting with the analyte in the sample to produce (either directly orindirectly) a compound that is characteristically absorptive at awavelength other than a wavelength at which the assay mediumsubstantially absorbs.

[0034] Polyamide matrices are particularly useful for carrying outreactions in which a substrate (analyte) reacts with an oxygen-utilizingoxidase enzyme in such a manner that a product is produced that furtherreacts with a dye intermediate to either directly or indirectly form adye which absorbs in a predetermined wavelength range. For example, anoxidase enzyme can oxidize a substrate and produce hydrogen peroxide asa reaction product. The hydrogen peroxide can then react with a dyeintermediate or precursor, in a catalysed or uncatalysed reaction, toproduce an oxidized form of the intermediate or precursor. This oxidizedmaterial may produce the colored product or react with a secondprecursor to form the final dye.

[0035] Nonlimiting examples of analyses and typical reagents include thefollowing materials shown in the following list. Analyte and Sample TypeReagents Glucose in blood, serum, Glucose Oxidase, Peroxidase urine orother biological and an Oxygen Acceptor fluids, wine, fruit juicesOxygen Acceptors include: or other colored aqueous O-dianisidine (1)fluids. Whole blood is a O-toluidine particularly preferred O-tolidine(1) sample typed. Benzidine (1) 2,2′-Azinodi-(3-ethylbenzthiazolinesulphonic acid-(6)) (1) 3-Methyl-2-benzothiazolinone hydrazone plusN,N-dimethylaniline (1) Phenol plus 4-aminophenazone (1) Sulfonated2,4-dichlorophenol plus 4-amino-phenazone (2)3-Methyl-2-benzothiazolinone hydrazone plus 3-(dimethylamino)benzoicacid (3) 2-Methoxy-4-allyl phenol (4) 4-Aminoantipyrine- dimethylaniline(5)

The Analysis Method

[0036] The analysis method of this invention relies on a change inabsorbance, as measured by diffuse reflectance, which is dependent uponthe amount of analyte present in a sample being tested. This change maybe determined by measuring the change in the absorbance of the testsample between two or more points in time.

[0037] The first step of the assay to be considered will be applicationof the sample to the matrix. In practice, an analysis could be carriedout as follows: First a sample of aqueous fluid containing an analyte isobtained. Blood may be obtained by a finger stick, for example. Anexcess over matrix saturation in the area where reflectance will bemeasured (i.e., about 5-10 microliters) of this fluid is applied to thereagent element or elements of the test device. Simultaneous starting ofa timer is not required (as is commonly required in the prior art), aswill become clear below. Excess fluid can be removed, such as by lightblotting, but such removal is also not required. The test device istypically mounted in an instrument for reading light absorbance; e.g.,color intensity, by reflectance, prior to application of the sample.Absorbance is measured at certain points in time after application ofthe sample. Absorbance refers in this application not only to lightwithin the visual wavelength range but also outside the visualwavelength range, such as infrared and ultraviolet radiation. From thesemeasurements of absorbance a rate of color development can be calibratedin terms of analyte level.

The Measuring Instrument

[0038] A suitable instrument, such as a diffuse reflectancespectrophotometer with appropriate software, can be made toautomatically read reflectance at certain points in time, calculate rateof reflectance change, and, using calibration factors, output the levelof analyte in the aqueous fluid. Such a device is schematically shown inFIG. 2 wherein a test device of the invention comprising backing 12 towhich reagent pad 11 is affixed is shown. Light source 5, for example ahigh intensity light emitting diode (LED) projects a beam of light ontothe reagent pad. A substantial portion (at least 25%, preferably atleast 35%, and more preferably at least 50%, in the absence of reactionproduct) of this light is diffusively reflected from the reagent pad andis detected by light detector 6, for example a phototransistor thatproduces an output current proportional to the light it receives. Lightsources 5 and/or detector 6 can be adapted to generate or respond to aparticular is wavelength light, if desired. The output of detector 6 ispassed to amplifier 7, for example, a linear integrated circuit whichconverts the phototransistor current to a voltage. The output ofamplifier 7 can be fed to track and hold circuit 8. This is acombination linear/digital integrated circuit which tracks or followsthe analog voltage from amplifier 7 and, upon command frommicroprocessor 20, locks or holds the voltage at its level at that time.Analog-to-digital converter 19 takes the analog voltage from track andhold circuit 8 and converts it to, for example, a twelve-bit binarydigital number upon command of microprocessor 20. Microprocessor 20 canbe a digital integrated circuit. It serves the following controlfunctions: 1) timing for the entire system; 2) reading of the output ofanalog/digital converter 19; 3) together with program and data memory21, storing data corresponding to the reflectance measured at specifiedtime intervals; 4) calculating analyte levels from the storedreflectances; and 5) outputing analyte concentration data to display 22.Memory 21 can be a digital integrated circuit which stores data and themicro-processor operating program. Reporting device 22 can take varioushard copy and soft copy forms. Usually it is a visual display, such as aliquid crystal or LED display, but it can also be a tape printer,audible signal, or the like. The instrument also can include astart-stop switch and can provide an audible or visible time output toindicate times for applying samples, taking readings, etc., if desired.

Reflectance Switching

[0039] In the present invention, the reflectance circuit itself can beused to initiate timing by measuring a drop in reflectance that occurswhen the aqueous portion of the suspension solution applied to thereagent pad (e.g., blood) migrates to the surface at which reflectanceis being measured. Typically, the measuring device is turned on in a“ready” mode in which reflectance readings are automatically made atclosely spaced intervals (typically about 0.2 seconds) from thetypically off-white, substantially dry, unreacted reagent strip. Theinitial measurement is typically made prior to penetration of the matrixby fluid being analyzed but can be made after the fluid has been appliedto a location on the reagent element other than where reflectance isbeing measured. The reflectance value is evaluated by themicroprocessor, typically by storing successive values in memory andthen comparing each value with the initial unreacted value. When theaqueous solution penetrates the reagent matrix, the drop in reflectancesignals the start of the measuring time interval. Drops in reflectanceof 5-50% can be used to initiate timing, typically a drop of about 10%.In this simple way there is exact synchronization of assay mediumreaching the surface from which measurements are taken and initiation ofthe sequence of readings, with no requirement of activity by the user.

[0040] Although the total systems described in this application areparticularly directed to the use of polyamide matrices and particularlyto the use of such matrices in determining the concentration of varioussugars, such as glucose, and other materials of biological origin, thereis no need to limit the reflectance switching aspect of the invention tosuch matrices. For example, the matrix used with reflectance switchingmay be formed from any water insoluble hydrophilic material and anyother type of reflectance assay.

Particular Application to Glucose Assay

[0041] A particular example with regard to detecting glucose in thepresence of red blood cells will now be given in order that greaterdetail and particular advantage can be pointed out. Although thisrepresents a preferred embodiment of the present invention, theinvention is not limited to the detection of glucose in blood.

[0042] The use of polyamide surfaces to form the reagent elementprovides a number of desirable characteristics in the present invention.These are that the reagent element is hydrophilic (i.e., takes upreagent and sample readily), does not deform on wetting (so as toprovide a flat surface for reflectance reading), is compatible withenzymes (in order to impart good shelf stability), takes up a limitedsample volume per unit volume of membrane (necessary in order todemonstrate an extended dynamic range of measurements), and showssufficient wet strength to allow for routine manufacture.

[0043] In a typical configuration, the method is carried out using anapparatus consisting of a plastic holder and the reagent element (thematrix having the signal producing system impregnated therein). Thepreferred matrix for use in preparing the reagent element is a nylonmicrofiltration membrane, particularly membranes made from nylon-66 caston a core of non-woven polyester fibers. Numerous nylon microfiltrationmembranes of this class are produced commercially by the Pall UltrafineFiltration Corporation having average pore sizes from 0.1 to 3.0microns. These materials show mechanical strength and flexibility,dimensional stability upon exposure to water, and rapid wetting.

[0044] Many variations in specific chemical structure of the nylon arepossible. These include unfunctionalized nylon-66 with charged endgroups (sold under the trademark Ultipore by Pall Ultrafine FiltrationCorporation; “Pall”). Positive charges predominate below pH 6 whilenegative charges predominate above pH 6. In other membranes the nylon isfunctionalized before the membrane is formed to give membranes withdifferent properties. Nylons functionalized with carboxy groups arenegatively charged over a wide pH range (sold as Carboxydyne by Pall).Nylons can also be functionalized with a high density of positivelycharged groups on its surface, typically quaternary amine groups, sothat they display little variation in charge over a wide pH range (soldas Posidyne by Pall). Such materials are particularly well suited forthe practice of the present invention. It is also possible to usemembranes having reactive functional groups designed for covalentimmobilization of proteins (sold as Biodyne Immuno Affinity membranes byPall). Such materials can be used to covalently attach proteins, e.g.enzymes, used as reagents. Although all of these materials are usable,nylon having a high density of positively charged groups on its surfaceprovide the best stability of reagents when formulated into a dryreagent pad. Unfunctionalized nylon gives the next best stability withthe carboxylated nylons next best.

[0045] Desirable results can be obtained with pore sizes ranging fromabout 0.2-2.0 μm, preferably about 0.5-1.2 μm, and most preferably about0.8 μm, when used with whole blood.

[0046] The form of the handle on which the reagent element is assembledis relatively unimportant as long as the handle allows access to oneside of the reagent element by sample and to the other side of thereagent element by incident light whose reflectance is being measured.The handle also aids in inserting the reagent element into theabsorbance measuring device so that it registers with the opticalsystem. One example of a suitable handle is a mylar or other plasticstrip to which a transfer adhesive such as 3M 465 or Y9460 transferadhesive has been applied. A hole is punched into the plastic throughthe transfer adhesive. A reagent element, typically in the form of athin pad, either containing reagents or to which reagents will later beadded, is then applied to the handle by means of the transfer adhesiveso that it is firmly attached to the handle in the area surrounding thehole that has been punched through the handle and the transfer adhesive.Such a device is illustrated in FIG. 1, which shows reagent pad 11attached to a Mylar handle 12 by means of adhesive 13. Hole 14 allowsaccess of the sample or incident light to one side of reagent pad 11while access to the other side of the reagent pad is unrestricted. Alldimensions of the reagent pad and handle can be selected so that thereagent pad fits securely into a reflectance-reading instrument inproximal location to a light source and a reflected-light detector.

[0047] If a nylon matrix is selected to form the reagent pad, when theindicated thicknesses are employed, it is preferred to have the reagentpad supported by the holder in such a manner that no more than 6mm,measured in any direction, is unsupported by the holder at the locationwere the sample is applied and light reflectance is measured. Largerunsupported areas tend to provide inadequate dimensional stability tothe membrane so that measurement of reflectance from the surface isadversely affected. A 5 mm diameter hole 14 in the reagent strip shownin FIG. 1 works quite satisfactorily.

[0048] There is no particular limit on the minimum diameter of such ahole, although diameters of at least 2 mm are preferred for ease ofmanufacture, sample application, and light reflectance reading.

[0049] Although a number of dyes could be used as indicators, the choicewill depend upon the nature of the sample. It is necessary to select adye having an absorbance at a wavelength different from the wavelengthat which red blood cells absorb light, with whole blood as the assaymedium, or other contaminants in the solution being analyzed with otherassay media. The MBTH-DMAB dye couple (3-methyl-2-benzothiazolinonehydrazone hydrochloride and 3-dimethylaminobenzoic acid), although beingpreviously described as suitable for color development for peroxidaselabels in enzyme immunoassays, has never been used in a commercialglucose measuring reagent. This dye couple gives greater dynamic rangeand shows improved enzymatic stability as compared to traditional dyesused for glucose measurement, such as benzidine derivatives.Furthermore, the MBTH-DMAB dye couple is not carcinogenic, acharacteristic of most benzidine derivatives.

[0050] Another dye couple that can be used in the r) measurement ofglucose is the AAP-CTA (4-aminoantipyrene and chromotropic acid) couple.Although this couple does not provide as broad a dynamic range asMBTH-DMAB, it is stable and suitable for use in the practice of thepresent invention when measuring glucose. Again, the AAP-CTA dye coupleprovides an expanded dynamic range and greater enzymatic activitystability than the more widely used benzidine dyes.

[0051] The use of the MBTH-DMAB couple allows for correction ofhematocrit and degree of oxygenation of blood with a single correctionfactor. The more typically used benzidine dyes do not permit such acorrection. The dye forms a chromophore that absorbs at approximately635 nm but not to any significant extent at 700nm. Slight variations inmeasuring wavelengths (±about 10 nm) are permitted. At 700 nm bothhematocrit and degree of oxygenation can be measured by measuring bloodcolor. Furthermore, light emitting diodes (LED) are commerciallyavailable for both 635 nm and 700 nm measurements, thereby simplifyingmass-production of a device. By using the preferred membrane pore sizedescribed above and the subject reagent formulation both hematocrit andoxygenation behavior can be corrected by measuring at the single 700 nmwavelength.

[0052] Two additional conditions were found to provide particularstability and long shelf life for a glucose oxidase/peroxidaseformulation on a polyamide matrix. These use a pH in the range of 3.8 to5.0, preferably about 3.8 to 4.3, most preferably about 4.0, and use ofa concentrated buffer system for applying the reagents to the matrix.The most effective buffer was found to be 10 weight percent citratebuffer, with concentrations of from 5-15% being effective These areweight/volume percentages of the solution in which the reagents areapplied to the matrix. Other buffers can be used on the same molarbasis. Greatest stability was achieved using a low pH, preferably aboutpH 4, an MBTH-DMAB dye system, and a high enzyme concentration ofapproximately 500-1000 U/ml of application solution.

[0053] In preparing the MBTH-DMAB reagent and the enzyme system thatforms the remainder of the signal producing system, it is not necessaryto maintain exact volumes and ratios although the suggested values belowgive good results. Reagents are readily absorbed by the matrix pad whenthe glucose oxidase is present in a solution at about 27-54% by volume,the peroxidase is present at a concentration of about 2.7-5.4 mg/ml,MBTH is present at a concentration of about 4-8 mg/ml, and DMAB ispresent at a concentration of about 8-16 mg/ml. The DMAB-MBTH weightratio is preferably maintained in the range of (1-4):1, preferably about(1.5-2.5):1, most preferably about 2:1.

[0054] The basic manufacturing techniques for the reagent element are,once established, straightforward. The membrane itself is strong andstable, particularly when a nylon membrane of the preferred embodimentis selected. Only two solutions are necessary for applying reagent, andthese solutions are both readily formulated and stable. The firstgenerally contains the dye components and the second generally containsthe enzymes. When using the MBTH-DMAB dye couple, for example, theindividual dyes are dissolved in an aqueous organic solvent, typically a1:1 mixture of acetonitrile and water. The matrix is dipped into thesolution, excess liquid is removed by blotting, and the matrix is thendried, typically at 50-60° C. for 10-20 minutes. The matrix containingthe dyes is then dipped into an aqueous solution containing the enzymes.A typical formulation would contain the peroxidase and glucose oxidaseenzymes as well as any desired buffer, preservative, stabilizer, or thelike. The matrix is then blotted to remove excess liquid and dried asbefore. A typical formulation for the glucose reagent is as follows:

Dye Dip

[0055] Combine:

[0056] 40 mg MBTH,

[0057] 80 mg DMAB,

[0058] 5 ml acetonitrile, and

[0059] 5 ml water.

[0060] Stir until all solids are dissolved and pour onto a glass plateor other flat surface. Dip a piece of Posidyne membrane (Pall Co.), blotoff excess liquid, and dry at 56° C. for 15 minutes.

Enzyme Dip

[0061] Combine:

[0062] 6 ml water,

[0063] 10 mg EDTA, disodium salt,

[0064] 200 mg Poly Pep, low viscosity, ≮Sigma

[0065] 0.668 g sodium citrate,

[0066] 0.523 g citric acid,

[0067] 2.0 ml 6 wt % Gantrez AN-139 dissolved in water GAF

[0068] 30 mg horseradish peroxidase, 100 units/mg, and

[0069] 3.0 ml glucose oxidase, 2000 units/ml.

[0070] Stir until all solids are dissolved and pour onto a glass plateor other flat surface. Dip a piece of membrane previously impregnatedwith dyes, blot off excess liquid, and dry at 56° C. for 15 minutes.

[0071] The electronic apparatus used to make the reflectance readingsminimally contains a light source, a reflected light detector, anamplifier, an analog to digital converter, a microprocessor with memoryand program, and a display device.

[0072] The light source typically consists of a light emitting diode(LED). Although it is possible to use a polychromic light source and alight detector capable of measuring at two different wavelengths, apreferred apparatus would contain two LED sources or a single diodecapable of emitting two distinct wavelengths of light. Commerciallyavailable LEDs producing the wavelengths of light described as beingpreferred in the present specification include a Hewlett PackardHLMP-1340 with an emission maximum at 635 nm and a Hewlett PackardQEMT-1045 with a narrow-band emission maximum at 700 nm. Suitablecommercially available light detectors include a Hammamatsu 5874-18K anda Litronix BPX-65.

[0073] Although other methods of taking measurements are feasible, thefollowing method has provided the desired results. Readings are taken bythe photo-detector at specified intervals after timing is initiated. The635 nm LED is powered only during a brief measuring time span thatbegins approximately 20 seconds after the start time as indicated byreflectance switching. If this reading indicates that a high level ofglucose is present in the sample, a 30 second reading is taken and usedin the final calculation in order to improve accuracy. Typically, highlevels are considered to begin at about 250 mg/dl. The background iscorrected with a 700 nm D) reading taken about 15 seconds after thestart of the measurement period. The reading from the photodetector isintegrated over the interval while the appropriate LED is activated,which is typically less than one second. The raw reflectance readingsare then used for calculations performed by the microprocessor after thesignal has been amplified and converted to a digital signal. Numerousmicroprocessors can be used to carry out the calculation. An AIM65single-board microcomputer manufactured by Rockwell International hasproven to be satisfactory.

[0074] The present methods and apparatuses allow a very simple procedurewith minimum operational steps on the part of the user. In use, thereagent strip Is placed in the detector so that the hole in the stripregisters with the optics of the detecting system. A removable cap orother cover is placed over the optics and strip to shield the assemblyfrom ambient light. The measurement sequence is then initiated bypressing a button on the measuring apparatus that activates themicrocomputer to take a measurement of reflected light from theunreacted reagent pad, called an R_(dry) reading. The cap is thenremoved and a drop of blood is applied to the reagent pad, typicallywhile the reagent pad is registered with the optics and the readingdevice. It is preferred that the reagent strip be left in register withthe optics in order to minimize handling. The instrument is capable ofsensing the application of blood or other sample by a decrease in thereflectance when the sample passes through the matrix and reflectedlight is measured on the opposite side. The decrease in reflectanceinitiates a timing sequence which is described in detail at otherlocations in this specification. The cover should be replaced within 15seconds of sample application, although this time may vary depending onthe type of sample being measured. Results are typically displayed atapproximately 30 seconds after blood application when a blood glucosesample is being measured, although a 20 second reaction is permissiblefor glucose samples having a concentration of glucose of less than 250mg/dl. If other samples are being measured, suitable times fordisplaying the result may differ and can be readily determined from thecharacteristics of the reagent/sample selected.

[0075] A particularly accurate evaluation of glucose level (or any otheranalyte being measured) can be made using the background current, i.e.,the current from the photodetector with power on but with no lightreflected from the reagent pad, in order to make a backgroundcorrection. It has been demonstrated that over a 2-3 month period thatthis value does not change for a particular instrument preparedaccording to the preferred embodiments of this specification, and it ispossible to program this background reading into the computer memory asa constant. With a slight modification of the procedure, however, thisvalue can be measured with each analysis for more accurate results. Inthe modified procedure the meter would be turned on with the lid closedbefore the reagent strip is in place, and the background current wouldbe measured. The test strip would then be inserted into the meter withthe cover closed, an R_(dry) measurement taken, and the procedurecontinued as described above. With this modified procedure thebackground current does not need to be stable throughout the life of themeter, thereby providing more accurate results.

[0076] The raw data necessary for calculating a result in a glucoseassay are a background current reported as background reflectance,R_(b), as described above; a reading of the unreacted test strip.R_(dry), also described above; and an endpoint measurement. Using thepreferred embodiments described herein, the endpoint is not particularlystable and must be precisely timed from the initial application ofblood. However, the meter as described herein performs this timingautomatically. For glucose concentrations below 250 mg/dl, a suitablystable endpoint is reached in 20 seconds, and a final reflectance, R₂₀,is taken. For glucose concentrations up to 450 mg/dl, a 30-secondreflectance reading, R_(30,) is adequate. Although the system describedherein displays good differentiation up to 800 mg/dl of glucose, themeasurement is somewhat noisy and inaccurate above 450 mg/dl, althoughnot so great as to cause a significant problem. Longer reaction timesshould provide more suitable readings for the higher levels of glucoseconcentration.

[0077] The 700 nm reflectance reading for the dual wavelengthmeasurement is typically taken at 15 seconds (R₁₅). By this time bloodwill have completely saturated the reagent pad. Beyond 15 seconds thedye reaction continues to take place and is sensed, to a small part, bya 700 nm reading. Accordingly, since dye absorption by the 700 nm signalis a disadvantage, readings beyond 15 seconds are ignored in thecalculations.

[0078] The raw data described above are used to calculate parametersproportional to glucose concentration which can be more easilyvisualized than reflectance measurements. A logarithmic transformationof reflectance analogous to the relationship between absorbtivity andanalyte concentration observed in transmission spectroscopy (Beer's Law)can be used if desired. A simplification of the Kubelka-Monk equations,derived specifically for reflectance spectroscopy, have provenparticularly useful. In this derivation K/S is related to analyteconcentration with K/S defined by Equation 1.

K/S-t=(1−R*t)²/(2×R*t)   (1)

[0079] R*t is the reflectivity taken at a particular endpoint time, t,and is the absorbed fraction of the incident light beam described byEquation 2, where R_(t) is the endpoint reflectance, R₂₀ or R₃₀.

R*t=(R _(t) −R _(b))/(R _(dry) −R _(b))   (2)

[0080] R*t varies from 0 for no reflected light (R_(b)) to 1 for totalreflected light (R_(dry)). The use of reflectivity in the calculationsgreatly simplifies meter design as a highly stable source and adetection circuit become unnecessary since these components aremonitored with each R_(dry) and R_(b) measurement.

[0081] For a single wavelength reading K/S can be calculated at 20seconds (K/S-20) or 30 seconds (K/S-30). The calibration curves relatingthese parameters to YSI (Yellow Springs Instruments) glucosemeasurements can be precisely described by the third order polynomialequation outlined in Equation 3.

YSI=a ₀ +a ₂(K/S)+a ₂(K/S)² +a ₃(K/S)³   (3)

[0082] The coefficients for these polynomials are listed in Table 1.TABLE 1 Coefficients for Third Order Polynomial Fit of Single WavelengthCalibration Curves K/S-20 K/S-30 a₀ −55.75 −55.25 a₁ 0.1632 0.1334 a₂−5.765 × 10⁻⁵ −2.241 × 10⁻⁵ a₃  2.58 × 10⁻⁸  1.20 × 10⁻⁸

[0083] The single chemical species being measured in the preferredembodiments is the MBTH-DMAB indamine dye 30 and the complex matrixbeing analyzed is whole blood distributed on a 0.8Posidyne membrane. Areview entitled “Application of Near Infra Red Spectrophotometry to theNondestructive Analysis of Foods: A Review of Experimental Results”, CRCCritical Reviews in Food Science and Nutrition, 18 (3) 203-30 (1983),describes the use of instruments based on the measurement of an opticaldensity difference ΔOD (λ_(a)-λ_(b)) where ODλ_(a) is the opticaldensity of the wavelength corresponding to the absorption maximum of acomponent to be determined and ODλ_(b) is the optical density at awavelength where the same component does not absorb significantly.

[0084] The algorithm for dual wavelength measurement is by necessitymore complex than for single wavelength measurement but is much morepowerful. The first order correction applied by the 700 nm reading is asimple subtraction of background color due to blood. In order to makethis correction, a relationship between absorbance at 635 nm and 700 nmdue to blood color can be and was determined by measuring blood sampleswith 0 mg/dl glucose over a wide range of blood color. The color rangewas constructed by varying hematocrit, and fairly linear relationshipswere observed. From these lines the K/S-15 at 700 nm was normalized togive equivalence to the K/S-30 at 635 nm. This relationship is reportedin Equation 4, where K/S-15 n is the normalized K/S-15 at 700 nm.

K/S-15n=(K/S-15×1.54)−0.133   (4)

[0085] Note that the equivalence of the normalized 700 nm signal and the635 nm signal is only true at zero 25 glucose. The expressions fromwhich the calibration curves were derived are defined by Equations 5 and6.

K/S-20/15=(K/S-20)−(K/S-15n)   (5)

K/S-30/15=(K/S-30)−(K/S-15n)   (6)

[0086] These curves are best fit by fourth-order polynomial equationssimilar to Equation 3 to which a fourth-order term in K/S is added. Thecomputer-fit coefficients for these equations are listed in Table 2.TABLE 2 Coefficients for Fourth-Order Polynomial Fit of Dual WavelengthCalibration Curves K/S-20/15 K/S-30/15 a₀ −0.1388 1.099 a₁ 0.10640.05235 a₂ 6.259 × 10⁻⁵ 1.229 × 10⁻⁴ a₃ −6.12 × 10⁻⁸ −5.83 × 10⁻⁸ a₄ 3.21 × 10⁻¹¹  1.30 × 10⁻¹¹

[0087] A second order correction to eliminate errors due tochromatography effects has also been developed. Low hematocrit sampleshave characteristically low 700 nm readings compared to higherhematocrit samples with the same 635 nm reading. When the ratio of(K/S-30)/(K/S-15) is plotted versus K/S-30 over a wide range ofhematocrits and glucose concentrations, the 20resulting line on thegraph indicates the border between samples which display chromatographyeffects (above the curve) and those that do not (below the curve). TheK/S-30 for the samples above the curve are corrected by elevating thereading to correspond to a point on the curve with the same(K/S-30)/(K/S-15).

[0088] The correction factors reported above were tailor made to fit asingle instrument and a limited number of reagent preparations. Thealgorithm can be optimized for an individual instrument and reagent inthe same manner that is described above.

[0089] In summary, the system of the present invention minimizesoperator actions and provides numerous advantages over prior artreflectance-reading methods. When compared to prior methods fordetermining glucose in blood, for example, there are several apparentadvantages. First, the amount of sample required to saturate the thinreagent pad is small (typically 5-10 microliters). Second, operator timerequired is only that necessary to apply the sample to the thinhydrophilic layer and close the cover (typically 4-7 seconds). Third, nosimultaneous timing start is required. Fourth, whole blood can be used.The method does not require any separation or utilization ofred-cell-free samples and likewise can be used with other deeply coloredsamples.

[0090] Several unobvious advantages arise as a result of the practice ofthe present invention with whole blood. Normally, aqueous solutions(like blood) will penetrate a hydrophilic membrane to give a liquidlayer on the opposite side of the membrane, a surface that is then notsuited for a reflectance measurement. It has been discovered, however,that blood, apparently because of interactions of red blood cells andproteins in the blood with the matrix, will wet the polyamide matrixwithout having an excess liquid penetrate the porous matrix to interferewith the reflectance reading on the opposite side of the matrix.

[0091] Furthermore, the thin membranes used in the present inventionwould be expected when wet to transmit light and return only a weaksignal to the reflectance measuring device. Prior teachings havegenerally indicated that a reflective layer is necessary behind thematrix in order to reflect sufficient light. In other cases a white padhas been placed behind the reagent pad prior to color measurement. Inthe present case, neither a reflective layer or a white pad is required.In fact, the invention is typically carried out with a light-absorbingsurface behind the reagent element when incident light is impinged uponthe matrix. Using a light-absorbing surface behind the reagent element,coupled with measuring reflectance at two different wavelengths, allowsacceptable reflectance measurements to be obtained without removal ofexcess liquid from the matrix, thereby eliminating a step typicallyrequired by previous teachings.

[0092] The invention now being generally described, the same will bebetter understood by reference to the following specific examples whichare presented for purpose of illustration only and are not to beconsidered limiting of the invention unless so specified.

EXAMPLE I Reproducibility

[0093] One male blood sample (JG, hematocrit=45) was used to collect thereproducibility data set forth in Tables 3-5. TABLE 3 Reproducibility ofa Single Wavelength MPX System Average (mg/dl) S.D. (mg/dl) % C.V. YSI(mg/dl) 20 sec. 30 sec. 20 sec. 30 sec. 20 sec. 30 sec. 25 23.1 23.0 2.12.04 9.1 9.0 55 53.3 53.2 3.19 3.32 6.0 6.3 101 101 101 3.0 3.3 3.0 3.3326 326.6 327 13.3 9.8 4.1 3.0 501 503 17.1 3.4 690 675 28 4.15 810 81337 4.5

[0094] TABLE 4 Reproducibility of a Dual Wavelength MPX System Average(mg/dl) S.D. (mg/dl) % C.V. YSI (mg/dl) 20 sec. 30 sec. 20 sec. 30 sec.20 sec. 30 sec. 25 25 27 1.34 1.55 5.4 5.7 55 55 57.4 2.58 2.62 4.7 4.6101 101 101.5 2.55 2.18 2.5 2.1 326 332 330 15.0 7.1 4.5 2.1 501 50521.3 4.2 690 687 22.8 3.3 810 817 30.4 3.7

[0095] TABLE 5 Reproducibility at a 3.0 mm Diameter Aperture % C.V. YSI(mg/dl) 4.7 mm 3.0 mm 55-100 4.8 4.9 300 3.0 5.0 600 3.8 5.5 avg. 3.95.1

[0096] The blood was divided into aliquots and spiked with glucoseacross a range of 25-800 mg/dl. Twenty determinations were made at eachglucose test level from strips taken at random from a 500 strip sample(Lot FJ4-49B). The results of this study lead to the followingconclusions:

[0097] 1. Single vs. Dual Wavelength. The average C.V. for the 30-seconddual result was 3.7% vs. 4.8% for the 30-second single wavelengthresult, an improvement of 23% across a glucose range of 25-810 mg/dl.There was a 33% improvement in C.V. in the 25-326 mg/dl glucose range.Here the C.V. decreased from 5.4% to 3.6%, a significant improvement inthe most used range. The 20-second dual wavelength measurement gavesimilar improvements in C.V. compared to the single wavelengthmeasurement in the 25-325 mg/dl range (Tables 3 and 4).

[0098] 2. Dual Wavelength, 20 vs. 30-second Result: The average C.V. fora 20-second result in the 25-100 mg/dl range is nearly identical to the30-second reading, 4.2% vs. 4.1%. However, at 326 mg/dl the 30-secondreading has a C.V. of 2.1% and the 20-second result a C.V. of 4.5%. Aswas seen in the K/S-20 response curve, the slope begins to decreasesharply above 250 mg/dl. This lead to poor reproducibility at glucoselevels greater than 300 for the 20-second result. From thisreproducibility data the cutoff for the 20-second result is somewherebetween 100 and 326 mg/dl. A cutoff of 250 mg/dl was later determinedfrom the results of the recovery study set forth in Example II.

[0099] 3. Aperture Size: A smaller optics aperture size, 3.0 mm us. 50min., was investigated. Initial experimentation using a 10-reokucate,hand-dipped disk sample did show improved C.V.s with the 3.0 mmaperture, apparently because of easier registration with the systemoptics. However, when machine-made roll membrane was used, the averageC.V. (Table 5) of the larger aperture, 4.7 mm, was 3.9% us. an averageC.V. for the 3.0 mm aperture of 5.1%. This 30% increase in C.V. wasprobably due to the uneven surface of the roll membrane lot as discussedbelow.

EXAMPLE II Recovery

[0100] For comparison of the present method (MPX) against a typicalprior art method using a Yellow Springs Instrument Model 23A glucoseanalyser manufactured by Yellow Springs Instrument Co., Yellow Springs,Ohio (YSI), blood from 36 donors was tested. The donors were dividedequally between males and females and ranged in hematocrit from 35 to55%. The blood samples were used within 30 hours of collection, withlithium heparin as the anti-coagulant. Each blood sample was dividedinto aliquots and spiked with glucose to give 152 samples in the rangeof 0-700 mg/dl glucose. Each sample was tested in duplicate for a totalof 304 data points.

[0101] Response curves were constructed from these data and glucosevalues then calculated from the appropriate equation (Tables 1 and 2).These MPX glucose values were then plotted vs. the YSI values to givescattergrams.

[0102] Comparison of MPX Systems: For both the 20-second and 30-secondmeasurement times there is visually more scatter in thesingle-wavelength scattergrams than the dual-wavelength scattergrams.The 20-second reading becomes very scattered above 250 mg/dl but the30-second measurement does not have wide scatter until the glucose levelis k500 mg/dl.

[0103] These scattergrams were quantitated by determining the deviationsfrom YSI at various glucose ranges. The following results were obtained.TABLE 6 Accuracy of MPX from Recovery Data MPX Wave- Measurement S.D.(mg/dl) C.V. for Range* length Time (sec.) 0-50 50-250 250-450 450-700Single 20 ±5.6 7.2 14.5 — Single 30 ±6.9 7.1 8.8 10.2 Dual 20 ±2.3 5.312.8 — Dual 30 ±2.19 5.5 5.8  8.4

[0104] Note: These are inter method C.V.s.

[0105] a. The dual wavelength system gave C.V.s that ranged 30% lowerthan the single wavelength system.

[0106] b. The single wavelength system, from 0-50 mg/dl, showed a S.D.of ±6-7 mg/dl whereas the S.D. for a dual wavelength measurement wasonly ±2.2 mg/dl.

[0107] c. The cutoff for a 30-second MPX measurement is 250 mg/dl. Forthe 50-250 mg/dl range both the 20- and 30-second measurements gavesimilar inter-method C.V.s (approximately 7% for single wavelength, 5.5%for dual wavelength). However, in the 250-450 mg/dl range inter-methodC.V.s more than double for the 20-second reading to 14.5% for the singleand 12.8% for the dual wavelength.

[0108] d. The 30-second reading was unusable above 450 mg/dl for boththe single and dual wavelength measurement (C.V.s of 10.2 and 8.4%).

[0109] In conclusion, two MPX systems gave optimum quantitation in the0-450 mg/dl range.

[0110] 1. MPX 30 Dual: This dual wavelength system gave a 30-secondmeasurement time with a 95% confidence limit (defined as the probabilityof a measurement being within 2 S.D. of the YSI) of 11.3% (C.V.) for therange from 50-450 mg/dl (Table 7) and ±4.4 mg/dl (S.D.) for 0-50 mg/dl.

[0111] 2. MPX 30/20 Dual: This dual wavelength system gave a 20-secondmeasurement time in the 0-250 mg/dl range and a 30-second time for the250-450 range. The 95% confidence limits are nearly identical to the MPX30 Dual System (Table 7), 11.1% (C.V.) for 50-450 mg/dl and ±4.6 mg/dl(S.D. for 0-50 mg/dl). TABLE 7 Comparison of 95% Confidence Limits forMPX, GlucoScan Plus and Accu-Chek bG* Reagent Strips Measuring Range MPXSingle Wavelength MPX Dual Wavelength mg/dl 20 sec. 30 sec. 20 sec. 30sec. 0-50 11.2 13.8 4.6 4.4 mg/dl mg/dl mg/dl mg/dl 50-250 14.4% 14.2%10.6% 11.0% 250-450  — 17.6% — 11.6% 77-405 GlucoScan Plus (DrexlerClinical) 15.9% 77-405 Accu-Chek bG (Drexler Clinical) 10.7% 50-450 MPX20/30 Dual Hybrid 11.1% 50-450 MPX 30 Dual 11.3

EXAMPLE III Stability

[0112] Most of the bench-scale work carried out in optimizing stabilitywas completed using hand-dipped 0.8 Posidyne membrane disks. TheSpecific dye/enzyme formulation was set forth previously.

[0113] 1. Room Temperature Stability: This study attempted to chart anychange in response of the 0.8 Posidyne membrane reagent stored at 18-20°C. over silica gel desiccant. After 2.5 months there was no noticeablechange as measured by the response of a room temperature sample vs. theresponse of a sample stored at 5° C. Each scattergram represented aglucose range of 0-450 mg/dl.

[0114] 2. Stability at 37° C.: A 37° C. stability study using the samereagent as the RT study was carried out. The differences in glucosevalues of reagent stressed at 37° C. vs. RT reagent, for strips stressedwith and without adhesive, was plotted over time. Although the data wasnoisy, due to the poor reproducibility of handmade strips, the stabilitywas excellent for strips whether they were stressed with or withoutadhesive.

[0115] 3. Stability at 56° C.: Eight 5- to 6-day stability studies werecarried out using different preparations of a similar formulation ondisk membrane (Table 8). For the low glucose test level (80-100 mg/dl)the average gulcose value dropped upon stressing by 3.4%, with thehighest drop being 9.55%. At the high test level (280-320 mg/dl) theglucose reading declined by an average of 3.4%, the largest declinebeing 10.0%. TABLE 8 Stability at pH = 4.0, Posidyne Disk ReagentFormulation Stressed for 5 to 6 Days at 56° C. % Difference (56° C. vs.RT Sample) Sample No. YSI (80-100 mg/dl) YSI (280-320 mg/dl) FJ22B −6.25+5.4 FJ27A −4.0 −5.14 FJ28B −2.4 −5.3 FJ30H −9.55 −10.0 FJ31C +4.43−1.24 FJ36 −3.2 −8.5 FJ48B* −3.0 −0.0 GM48A* −3.0 −2.5 Average of 8 −3.4−3.4

[0116] A study of the 56° C. stressing of this membrane over a 19-dayperiod showed no major difference for strips stressed with or withoutadhesive. In both cases the 19-day decline in glucose value was <15% atlow test levels (80-100) and 300 mg/dl.

[0117] Another 56° C. study using hand-dipped 0.8 Posidyne membrane withtwice the normal concentration of enzyme and dye was completed. Twoseparate preparations of the same formulation were made up and thestability measured over a 14-day period. The average results of the twostudies were plotted. Changes were within ±10% over the 14-day period atboth the high and low glucose test level. These data show thisformulation to be particularly stable.

EXAMPLE IV Sample Size

[0118] The sample size requirements for MPX are demonstrated in Table 9.TABLE 9 Effect of Sample Size on MPX Measurements Sample Average Size(μl) Dual Wavelength Single Wavelength Low Glucose YSI = 56 3 41 50 3931 40 31 42 30 19 30 4 44 49 49 49 48 41 45 44 45 44 5 54 48 49 51 50 5049 48 49 49 10 48 48 50 47 48 54 53 56 55 54 20 49 49 49 50 49 55 57 5860 58 High Glucose YSI = 360 2 301 260 276 286 280 274 232 244 260 252 4383 378 367 341 367 361 356 342 318 344 5 398 402 382 370 388 378 387366 351 370 10 364 362 378 368 368 356 358 379 369 366 20 375 370 380378 376 380 382 389 385 384

[0119] The volumes reported in the table were transferred to the reagentpad shown in FIG. 1 using a micro pipet. When blood from a finger stickis applied to a strip the total sample cannot be transferred, thereforethe volumes reported here do not represent the total sample size neededto be squeezed from the finger for the analysis. A 3-μl sample is theminimum necessary to completely cover the reagent pad circle. This doesnot provide enough sample to completely saturate the reagent pad and MPXgives low results. A 4-μl sample barely saturates the reagent pad, whilea 5-μl sample is clearly adequate. A 10-μl sample is a large shiny dropand a 20-μl sample is a very large drop and is only likely to be usedwhen blood from a pipet is used for sampling.

[0120] At low glucose concentration the single wavelength result hassome dependence on sample size which is completely eliminated using thedual wavelength measurement. Although this dependence with the singlewavelength might be considered acceptable, it is clearly undesirable.

EXAMPLE V Reproducibility

[0121] Experimental measurements described above were always run inreplicate, usually 2, 3 or 4 determinations per data point. These setshave always shown close agreement even for samples with extremehematocrits or extreme oxygen levels. C.V.s were well below 5%. Itappears, therefore, that reproducibility is very good to excellent.

[0122] The subject invention provides for many advantages over systemswhich are presently available commercially or have been described in theliterature. The protocols are simple and require little technical skilland are relatively free of operator error. The assays can be carried outrapidly and use inexpensive and relatively harmless reagents, importantconsiderations for materials employed in the home. The user obtainsresults which can be understood and used in conjunction with maintenancetherapy. In addition, the reagents have long shelf lives, so that theresults obtained will be reliable for long periods of time. Theequipment is simple and reliable and substantially automatic.

[0123] All patents and other publications specifically identified inthis specification are indicative of the level of skill of those ofordinary skill in the art to which this invention pertains and areherein individually incorporated by reference to the same extent aswould occur if each reference were specifically and individuallyincorporated by reference.

[0124] The invention now being fully described, it will be apparent toone of ordinary skill in the art that many modifications and changes canbe made thereto without departing from the spirit or scope of theinvention as defined in the following claims.

What is claimed is:
 1. In a method for determining glucose in a bloodsample, employing a membrane and a signal-producing system that producesa light-absorptive dye product, said system being bound to the membrane,in which the amount of said dye product is determined by means of areflectance measurement from a surface of said membrane, an improvementwhich comprises: applying whole blood to a surface of said membrane, andmaking said measurement on a surface of said membrance other than thesurface to which said sample is applied.
 2. A method according to claim1, wherein said signal-producing system produces a dye product whichabsorbs light at a wavelength different from the wavelength at whichsaid red blood cells absorb and said reflectance measurement is made attwo different wave lengths, one at the absorption wavelength of the redblood cells and one at the absorption wavelength of said dye product. 3.A method according to claim 2, wherein said two wavelengths are at about635 and 700 nm.
 4. A method according to claim 1, wherein said signalproducing system comprises glucose oxidase, peroxidase and MBTH-DMABindicator.
 5. A method according to claim 1, wherein the surface of saidmembrane is a polyamide.
 6. A method according to claim 5, wherein saidpolyamide is positively charged and glucose oxidase, peroxidase andMBTH-DMAB indicator are bound to said membranes prior to use in saidmethod.
 7. A method for determining glucose comprising the steps of:applying a whole blood sample to a reagent element, wherein said reagentelement comprises a hydrophilic matrix to which is bound glucoseoxidase, peroxidase, and a dye indicator capable of forming a reactionproduct; allowing the sample to penetrate said membrane; initiatingtiming of a predetermined timed reflectance readings by detectingpenetration of the sample through said membrane; and by a decrease inreflectance; and determining the amount of glucose in said a sample bythe additional change in reflectance resulting from absorbance by saiddye product.
 8. A method according to claim 7, wherein said dyeindicator is indicator.
 9. A method according to claim 7, wherein saidhydrophilic memberane has a polyamide surface.
 10. A method according toclaim 9, wherein said hydrophilic membrane is positively charged.
 11. Amethod of initiating timing of a measurement in a reflectance-readingdevice, which comprises: taking at least one first reflectance readingfrom a first surface of a porous matrix prior to application of a sampleto a second surface of said matrix; taking at least one additionalreflectance reading from said first surface; comparing said additionalreflectance reading to said first reflectance reading and initiatingtime measurement upon a predetermined drop in reflectance resulting fromsaid sample reaching said first surface; and taking at least onemeasurement reflectance reading at a predetermined time after saidadditional reflectance reading differs from said reflectance value bysaid predetermined difference.
 12. A method according to claim 11,wherein said surface is initially dry and said additional reflectancereading differs from said first reflectance value when a liquid assaymedium penetrates to said first surface of said matrix.
 13. A methodaccording to claim 12, wherein said matrix has a dye precursor bound tothe matrix.
 14. A measuring apparatus for the determination of ananalyte in a fluid, which comprises: a container having light-tightclosure adapted to removably contain a porous matrix capable ofcontaining an absorbed fluid to be analyzed; means for illuminatingsurface of said matrix; means for detecting light of two differentwavelengths reflected from said matrix; control means for collectingreadings of reflected light and calculating a value for the amount ofanalyte in said fluid based on readings of analyte or reaction productabsorbance from the first wavelength of reflected light and ofbackground absorbance from the second wavelength of reflected light; andmeans for reporting said value.
 15. The apparatus of claim 14, whereinsaid apparatus further comprises a self-contained electronic powersupply operationally connected to said means for illuminating, means fordetecting, control means, and reporting means.
 16. The apparatus ofclaim 14, wherein said means for illuminating and means for detectinglight of two different wavelengths together comprise: 1) two lightsources of different wavelengths and a single light detector; or 2) apolychromic light source and two detectors restricted to detectingdifferent wavelengths of light.
 17. The apparatus of claim 14, whereinthe first wavelength of light is from 690 to 710 nm and the secondwavelength of light is from 625 to 645 nm.
 18. The apparatus of claim14, wherein said control means causes a timing circuit to be initiatedbased on initial decrease in reflectance of either wavelength of light,said decrease occuring when said fluid penetrates said matrix afterbeing applied to said matrix on a surface other than the surfaceilluminated by said illuminating means.
 19. The apparatus of claim 14,wherein said control means causes one or more readings of reflectedlight to be taken at predetermined intervals after said initial decreasein reflectance.
 20. The apparatus of claim 19, wherein said controlmeans is capable of collecting and storing a reading of reflectance fromsaid reagent pad prior to application of fluid to said reagent pad. 21.The apparatus of claim 20, wherein aid control means is further capableof collecting and storing a background detector current reading in theabsence of light reflected from said reagent pad.
 22. The apparatus ofclaim 21 wherein when power is supplied to said control means in ananalyte detection mode, said control means automatically collects andcompares sequential reflectance readings from said detector, initiates atiming circuit upon detection of a drop in reflectance, collectsreaction reflectance readings at predetermined intervals after said dropin reflectance, calculates a value for the concentration of analyte insaid fluid being analyzed from said reaction reflectance readings, andtransfers said value to said reporting means.
 23. The apparatus of claim20, wherein when power is supplied to said control means in a basereflectance mode prior to said analyte detection mode, said controlmeans collects and stores a base reflectance reading from said matrixprior to application of fluid to said matrix and wherein when power isnext supplied to said control means in the analyte detection mode, saidbase reflectance reading is subtracted from said reaction reflectancereadings by said control means prior to calculation by said controlmeans of said value.
 24. A method of determining analyte concentrationin a liquid, which comprises: quantitatively measuring base reflectancefrom a first surface of a reagent element comprising an inert porousmatrix and a reagent system capable of interacting with said analyte toproduce a light-absorbing reaction product, said reagent system beingimpregnated in the pores of said matrix, prior to application of saidliquid to said reagent element; quantitatively measuring reactionreflectance from said reagent element after application of said liquidto a second surface of said reagent element other than the first surfacefrom which said reaction reflectance measured and after said liquid hasmigrated through said reagent element from said second surface to saidfirst surface; quantitatively measuring interference reflectance fromsaid first surface of said reagent element after said application ofliquid using a wavelength of light different from the wavelength oflight used to measure said reaction reflectance; and calculating a valueexpressing said concentration from said reflectance measurements. 25.The method of claim 24, wherein said matrix has surfaces formed from apolyamide.
 26. The method of claim 24 wherein the average diameter ofthe pores in said matrix is from 0.2 to 1.0 m and said liquid is wholeblood.
 27. The method of claim 26, wherein said reagent produces alight-absorbing reaction product upon reacting with glucose.